In many types of cancer cells, differentiation antigens are expressed. These antigens have been used as targets in cancer therapy. For example, CD19, CD20, CD22 and CD25 have successfully been used as targets in hematopoietic malignancies (Press, et al., New Eng. J. Med. 329:1219–1224 (1993); and Osterborg, et al., J. Clin. Oncol. 15:1567–1574 (1997)). This targeted cancer therapy has not, however, been successful with solid tumors, in large part because the targeted antigens are also expressed in tissues from which the tumors arose. Thus, such targeted therapies kill healthy cells as well as the malignant cells.
In the United States, despite therapy, an estimated 15,000 women die of ovarian cancer each year. Although less common than ovarian cancer, mesotheliomas are known to be resistant to all chemotherapeutic agents and therefore have a high mortality rate. Because of the morbidity of these cancers, new therapeutic approaches to these malignancies are needed.
Common to ovarian, squamous cell and some stomach cancers as well as mesotheliomas is the expression of mesothelin on the cell surface (Chang, et al., Cancer Res. 52:181–186 (1992); Chang, et al., J. Surgical Pathology 16:259–268 (1992); and Chang, et al., Nat'l Acad. Sci. USA 93:136–140 (1996)). Mesothelin is a 40 kD GPI-linked glycoprotein antigen present on the surface of mesothelial cells. It is synthesized as a 69 kD precursor which is then proteolytically processed. The 30 kD amino terminus is secreted and has been termed megakaryocyte potentiating factor (Yamaguchi, et al., J. Biol. Chem. 269:805–808 (1994)). The 40 kD carboxyl terminus remains bound to the membrane as mature mesothelin (Chang, et al., Nat'l Acad. Sci. USA 93:136–140 (1996)). Unlike many cell surface antigens present on cancer cells, the membrane-bound form of mesothelin cannot be detected in the blood of cancer patients and is not shed by cultured cells into medium (Chang, et al., Cancer Res. 52:181–186 (1992)). In addition to malignant cells, mesothelin is also found on the cell surface of cells of mesothelial origin, including ovarian cancers. Because damage to cells in these tissues would not lead to life-threatening consequences, the presence of mesothelin on the surface of cancer cells makes it a promising candidate for targeted therapies.
Immunotoxins are antibodies directed against cell surface antigens joined to a toxic moiety. In the treatment of cancer, the antibody preferably is directed against a cell surface antigen expressed only on cancer cells. However, if the death of normal cells which also express the surface antigen is not more life-threatening than the existence of the malignancy, antibodies directed against cell surface antigens expressed on non-malignant cells can be used in cancer therapy. The toxic moiety of the immunotoxin can be any toxin that is not harmful to non-targeted cells at low concentrations after systemic administration. Such a toxin is the Pseudomonas aeruginosa exotoxin (PE). Previous studies with PE have demonstrated that the active portion of the protein is composed of domain II and III, both of which are located at the carboxyl end of the toxin. Other toxins under development or consideration for use as the toxic moiety of immunotoxins are diphtheria toxin (Watson et al., Intl J Cancer, 61:233–40 (1995)), saporin (e.g., Chandler et al., Intl J. Cancer 78(1):106–11 (1998)) pokeweed antiviral protein (e.g., Ek et al., Clin Cancer Res, 4(7):1641–7 (1998), ricin (e.g., Ohtomo et al., Anticancer Res, 18(6A):4311–5 (1998)), and bryodin 1 (e.g., Francisco et al., J Biol Chem, 272(39):24165–9 (1997)). For use as immunotoxins, often the natural toxin molecule is altered to avoid non-specific toxicity or other undesirable effects. For example, the A and B chains of ricin are usually separated so that the toxic effect of the A chain can be used while avoiding the non-specific binding which would otherwise be provided by the B chain.
The antibodies that target the immunotoxin can be polyclonal, monoclonal, or recombinant antibodies, such as chimeras or variable region fragments. If the antibody is non-recombinant, the immunotoxin must be formed by chemical conjugation of the antibody to the toxic moiety. If the antibody is produced recombinantly, the antibody can be joined to the toxin through chemical bonding or through recombinant fusion. In recombinant fusion, cDNA encoding the antibody is inserted, in frame, into a plasmid that already contains cDNA which encodes the toxin. Of course, the reverse could be done as well; the toxin cDNA can be inserted into a plasmid carrying cDNA which encodes the antibody.
Because of the potential large size of the immunotoxin, it is sometimes desired to join only a fragment of an antibody to the toxic moiety. Fab, Fab′ and F(ab)2 fragments can be made from polyclonal, monoclonal and chimeric antibodies and then joined to the toxin through chemical bonding.
Alternatively, a cDNA can be produced in which the variable regions of an antibody are connected to essential framework regions. These smaller antibodies are then secreted as double chain Fv antibodies or, if the heavy and light chain regions are joined either directly or through a peptide linker, as single chain Fv antibodies (scFv).
One method of creating a scFv is through phage display libraries made from splenic mRNA of mice immunized with an immunogen (Chowdhury, et al., Mol. Immunol. 34:9–20(1997)). If a protein immunogen is naturally found in mammals but is recombinantly expressed in prokaryotes, however, the protein will not have the correct glycosylation pattern and may not have the correct conformation. Antibodies developed by the mouse in response to this immunogen may not recognize the protein in its native state. One solution to this problem is to immunize animals with the native protein made in mammalian cells, but purification from mammalian cells of sufficient amounts of some proteins, in particular cell surface proteins, may not be possible. Another solution, although not as common, is to immunize animals with cDNA which encodes the immunogen. The cDNA, under the control of an appropriate promoter, is introduced into the animal. After boosting injections and when the antibody titer reaches a maximum, the animals are sacrificed and the spleens removed to create the phage display library.
Development of targeted therapies against mesothelin-expressing malignancies has been hampered by difficulties in developing high-affinity antibodies that are internalized well. As we noted in a recent publication, Chowdhury et al., Proc Natl Acad Sci USA, 95:669–674(1998) (hereafter, “Chowdhury 1998”), purification of sufficient amounts of mesothelin from mammalian cells has not been possible, and recombinant mesothelin expressed in Escherica coli has a low affinity for mesothelin-positive cells. We there reported that, by immunizing mice with plasmids containing DNA encoding mesothelin, we were able to elicit high titers of anti-mesothelin antibodies. We further reported that, using splenic RNA and phage display technology, we were able to isolate a single-chain Fv (“scFv”), which we called SS scFv, that binds with high affinity to mesothelin.
While SS appears to be a useful targeting agent for immunoconjugates, such as immunotoxins, even higher affinity antibodies are expected to improve the ability to detect mesothelin-expressing cancers, and to enhance the ability of immunotoxins to kill cancer cells expressing mesothelin. Accordingly, a need remains for improved means of generating antibodies with high affinity to target antigens. In particular, a need remains for higher affinity antibodies to mesothelin.